This proposal focuses on naturally occurring, biologically active small molecules - their isolation, their structural characterization, their ability to modulate biological responses, and ways of finding more of them. It is organized around three specific aims: 1) the discovery of new antibiotics, 2) the discovery of new signaling molecules, and 3) new technical approaches for discovering biologically active small molecules. 1. Antibiotics. The increasing spread of antibiotic resistance highlights both the necessity and urgency for new antibiotics. In addition, antibiotic assays are easily implemented surrogate assays for other kinds of biological activity such as antitumor agents. New chemotypes are most likely to be discovered from new sources, and this specific aim focuses on three different approaches: a) DNA-based approaches to discover antibiotic pathways in Pantoea agglomerans and from uncultured bacteria, b) screens for small molecule modulators of biofilm formation in pathogenic bacteria, and c) an in vivo antibiotic screen in a model organism. 2. Signaling molecules. Most small organisms - yeast, bacteria, and nematode worms for example - communicate using small molecule signals. Bacterial quorum sensors are the best known, but the catalog of these and other small signaling molecules is woefully incomplete. Some of the missing members could provide important insights into medically important processes such as pathogenesis and aging. Projects to find pheromones in the model organism Caenorhabditis elegans and the small molecule activators of global transcriptional regulators in the pathogenic bacteria Pseudomonas aeruginosa are presented. The emphasis on organisms with well-characterized genomes and genetic tools accelerates the study small molecule signal pathways, provides a rapid entry into biosynthesis, and allows the identification of related pathways in humans. 3. Biologically active small molecules from diverse natural sources - new technology. New biologically active small molecules are needed for many therapeutic categories. The proposal discusses: 1) investigating the feasibility of arraying prefractionated natural product extracts in a small molecule microarray format to speed up screening and 2) using NMR analysis of mixtures to identify culturing methods that lead to the production of previously cryptic molecules.